The bacterium Vibrio cholerae causes the severe diarrheal disease cholera, which remains a serious cause of morbidity in developing areas. For V. cholerae to cause disease, a collection of virulence genes, including the genes encoding cholera toxin and a toxin co-regulated pilus must be expressed appropriately. Virulence gene expression is regulated coordinately by a cascade of transcription factors. The ToxRS and TepsilonPH proteins activate transcription of another transcription activator, ToxT, which then directly activates transcription of over 20 virulence genes. The goals of this project are to understand the structure and function of ToxT. Studies using the infant mouse model will directly test the importance of appropriate temporal expression of virulence genes to colonization by utilizing a strain that expressed ToxT constitutively. The 4 ToxT-activated operons that have not been well characterized will also be studied both in vivo and in vitro. A consensus DNA binding site for ToxT will be determined by identifying and comparing sequences in genes ToxT is known to regulate, and by in vitro selection of DNA sequences to which ToxT binds with high affinity. Residues in ToxT specifically involved in transcription activation will be identified by a genetic screen/selection that isolates ToxT mutants defective in activation but able to bind DNA. Finally, since evidence exists that ToxT activity may be regulated, the mechanism for regulation will be investigated by measuring ToxT levels under non-inducing conditions, and searching for effectors of ToxT activity.